Controlled release microparticles

ABSTRACT

Formulations for controlled, sustained release of biologically active agents for the treatment of ocular disorders have been developed. These formulations are based on solid microparticles formed of the combination of biodegradable, synthetic polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and copolymers thereof. The microparticles are characterized by low burst levels and efficient drug loading and sustained release.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/741,741, filed Dec. 2, 2005, and U.S. Provisional ApplicationSer. No. 60/780,760, filed Mar. 9, 2006, and U.S. ProvisionalApplication Ser. No. 60/796,071, filed Apr. 28, 2006, all of which arehereby incorporated in its entirety by reference.

FIELD OF THE INVENTION

The invention relates to drug delivery. In particular, the inventionrelates to compositions and methods for the sustained delivery oftherapeutic agents using microparticles. More particularly, theinvention relates to sustained release microparticle compositions andmethods of use for ophthalmic administration.

BACKGROUND OF THE INVENTION

As new treatment modalities for ophthalmic diseases become available,the number of intravitreous injections administered is expected toincrease dramatically. For example, intravitreous injection of thevascular endothelial growth factor (VEGF) inhibitor, Macugen® ((OSI)Eyetech, Inc. NY, N.Y.), has become available for the treatment ofage-related macular degeneration. Macugen is currently delivered viaintravitreous injection every six weeks.

Advantages of intravitreous injection of medicines and diagnosticsinclude the achievement of maximum vitreous concentrations whileminimizing toxicity attributed to systemic administration. While theseadvantages are becoming widely appreciated, the ophthalmology communityturns its focus to various complications potentially associated withintravitreous injection. Risks of intravitreous injection, some visionthreatening, include endophthalmitis, retinal detachment,iritis/uveitis, inflammation, intraocular hemorrhage, ocularhypertension, hypotony, pneumatic retinopexy, and cataract (R. D. Jageret al., Retina 24:676-698, 2004 and C. N. Ta, Retina, 24:699-705, 2004).Methods of minimizing such risks include developing sustained releaseophthalmic formulations to minimize the number of intraocularinjections.

Ophthalmic inserts are solid devices intended to be placed in theconjunctival sac and to deliver the drug at a comparatively slow rate.One such device is Ocusert® (Alza Corporation, Mountain View, Calif.),which is a diffusion unit consisting of a drug reservoir enclosed by tworelease-controlling membranes made of a copolymer. M. F. Saettoneprovides a review of continued endeavors devoted to ocular delivery.(“Progress and Problems in Ophthalmic Drug Delivery”, Business Briefing:Pharmatech, Future Drug Delivery, 2002, 167-171). Other implantstrategies have been developed for small, highly potent, lipophilictherapeutics. (G. A. Peyman, et al., “Delivery Systems for IntraocularRoutes” Advanced Drug Delivery Reviews, (1995) 16, 107.) While theseimplants are effective for the delivery of steroids, the small size ofthe implants preclude long-term (>30 days) delivery of large,water-soluble compounds. In addition, formulation conditions for mostpolymeric delivery systems are not compatible with proteins, antibodies,and other biotherapeutics (S. P. Schwendeman et al., “Peptide, protein,and vaccine delivery from implantable polymeric systems: Progress andchallenges” Controlled Drug Delivery, (1997) 229).

Encapsulation of pharmaceuticals in biocompatible, biodegradable polymermicroparticles can prolong the maintenance of therapeutic drug levelsrelative to administration of the drug itself. Sustained release may beextended up to several months depending on the formulation and theactive molecule encapsulated. In order to prolong the existence at thetarget site, the drug may be formulated within a matrix into a slowrelease formulation (see, for example, Langer (1998) Nature, 392,Supplement, 5-10). Following administration, drug then is released viadiffusion out of, or via erosion of the matrix. Encapsulation withinbiocompatible, biodegradable polyesters, for example, copolymers oflactide and glycolide, has been utilized to deliver small moleculetherapeutics ranging from insoluble steroids to small peptides.Presently, there are over a dozen lactide/glycolide polymer formulationsin the marketplace, the majority of which are in the form ofmicroparticles (T. Tice, “Delivery with Depot Formulations” DrugDelivery Technology, (2004) 4(1)).

Several techniques for the production of lactide/glycolide polymermicroparticles containing biological or chemical agents by anemulsion-based manufacturing technique have been reported. In general,the methods include preparation of a first phase consisting of anorganic solvent, a polymer and a biological or chemical agent dissolvedor dispersed in the first solvent. A second phase comprises water and astabilizer and, optionally, the first solvent. The first and secondphases are emulsified and, after an emulsion is formed, the firstsolvent is removed from the emulsion, producing hardened microparticles.

Microparticles can also be produced using a water-in-oil-in-water(w/o/w) process. W/o/w emulsions can be considered as an aqueousemulsion of oil droplets which in turn contain a dispersed aqueousphase. Examples of w/o/w emulsion processes are described in U.S. Pat.Nos. 4,954,298; 5,330,767; 5,851,451 and 5,902,834, each of which arehereby incorporated herein by reference in their entirety. The w/o/wprocess described above is typically used for water-soluble molecules.

In addition, U.S. Pat. No. 6,706,289, hereby incorporated in itsentirety by reference, discloses controlled release formulations ofbiologically active molecules that are coupled to hydrophilic polymerssuch as polyethylene glycol and methods of their production. Theformulations are based on solid microparticles formed of the combinationof biodegradable, synthetic polymers such as poly(lactic acid) (PLA),poly(glycolic acid) (PGA), and copolymers thereof. PCT WO 03/092665,hereby incorporated in its entirety by reference, discloses microsphereformulations for the sustained delivery of an aptamer, for example, ananti-Vascular Endothelial Growth Factor aptamer, to a pre-selected locusin a mammal. Such formulations are further disclosed in K. G.Carrasquillo et al., “Controlled Delivery of the Anti-VEGF AptamerEYE001 with Poly(lactic-co-glycolic) Acid Microspheres,” I.O.V.S. (2003)44(1), 290.

Patient acceptance and safety are key issues that will play a role inwhich treatments are used. Frequent intraocular injections may not befavorable because they cause patient discomfort and sometimes fear,while risking permanent tissue damage. Therefore there remains a needfor developing sustained release ophthalmic formulations to minimize thenumber of intraocular injections.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for thesustained delivery of a biologically active agent using microparticles.In a particular aspect, the present invention provides sustained releasemicroparticle compositions and methods for ophthalmic administration.

In one aspect, the present invention provides a composition comprisingsustained release microparticles having the ability to be administeredby syringe to the eye. According to this aspect, the present inventionprovides microparticle formulations syringable through a 27-gauge needleor narrower (smaller).

In one embodiment, the microparticles release a biologically activeagent over a period of at least about 1-12 months. In a furtherembodiment, the microparticles release a biologically active agent overa period of at least about 3-6 months.

In another embodiment, the microparticles have a core load of at leastabout 10% by weight of the biologically active agent.

In another embodiment, the microparticles have an initial 24-hour invivo burst of less than about 10% by weight of the core load of thebiologically active agent.

In another aspect, the present invention provides compositions andmethods for the sustained delivery of an aptamer conjugated to ahydrophilic polymer such as polyethylene glycol. According to aparticular embodiment, the aptamer comprises pegaptanib.

In another aspect, the present invention provides compositions andmethods for the sustained delivery of an anti-VEGF agent. According to aparticular embodiment, the anti-VEGF agent comprises an aptamer.

In another aspect, the present invention provides a compositioncomprising sustained release microparticles comprising pegaptanib havingthe ability to be administered to a subject by a syringe via a 27-gaugeneedle or smaller.

In one embodiment, the microparticles release pegaptanib over a periodof at least about 1-12 months. In another embodiment, the microparticlesrelease pegaptanib over a period of at least about 3-6 months.

In another embodiment, the microparticles have a core load of at leastabout 10% by weight of pegaptanib.

In another embodiment, the microparticles have an initial 24-hour invivo burst of less than about 10% by weight of the core load ofpegaptanib.

In another aspect, the present invention provides methods ofadministering sustained release microsphere formulations to achieve adesired pharmacokinetic profile. According to one embodiment,microparticles comprising a biologically active agent are suspended in apharmaceutically acceptable solution and administered by syringe to theeye.

According to another embodiment, microparticles comprising abiologically active agent are suspended in a solution comprising thebiologically active agent and administered by syringe to the eye.Utilizing the vitreal residence time of the biologically active agent asa polymer clearance window allows the polymeric metabolites to becleared while sustaining a therapeutically relevant level within theeye.

The present invention has several advantages. In particular, themicroparticles of the present invention are easily suspendable andsyringable while able to provide increased duration, increasedstability, decreased burst and controlled, sustained or delayed releaseof biologically active molecules in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning electron micrograph showing the external morphologyof a sample of microspheres of the present invention.

FIG. 2 is a scanning electron micrograph of cross-sectionedpegaptanib-PLGA microspheres of the present invention having a low burstrelease.

FIG. 3 is a scanning electron micrograph showing the external morphologyof a sample of microspheres of the present invention formed by anwater-in-oil-in-water process.

FIG. 4 is a schematic representation illustrating an exemplaryoil-in-water process for forming microparticles of the presentinvention.

FIG. 5 is a schematic representation illustrating an exemplarywater-in-oil-in-water process for forming microparticles of the presentinvention.

FIG. 6 is a schematic representation illustrating an exemplary packedbed apparatus with various components according to an embodiment of thepresent invention.

FIG. 7 is a chart representing particle size distribution of typicalmicroparticles before sieving.

FIG. 8 shows a RP-HPLC chromatogram used to measure the core load andpurity of pegaptanib extracted from microspheres.

FIG. 9 is a graph depicting the release profiles of variouspegaptanib-PLGA microparticles formed by an oil-in-water process. Invitro dissolution date demonstrating control release kinetics fromPegaptanib-PLGA microspheres.

FIG. 10 is a graph depicting the release profiles of variouspegaptanib-PLGA microparticles formed by an water-in-oil-in-waterprocess. In vitro dissolution rate demonstrating control releasekinetics from Pegaptanib-PLGA microspheres.

FIG. 11 is a graph depicting the results of cell proliferation assays ofhuman umbilical vein endothelial cells (HUVEC) incubated with EYE001formulations after release from PLGA microparticles.

FIG. 12 is a graph depicting the results of 28 day in vivo releasestudy. The graph shows pegaptanib concentration in rabbits plasmasamples administered intravitreous with 5 mg of PLGA microparticlescontaining 15% weight percent pegaptanib.

FIG. 13 is a graph depicting the in vitro release profile of PLGApegaptanib microspheres suspended in PBS injection vehicle containing0.02% surfactant at a concentration of 100 mg microspheres permilliliter over an 86 day study.

FIG. 14 is a graph depicting the results of 86 day in vivo release studyin rabbits dosed intravitreously with 5 mg of PLGA microparticlescontaining 15% weight percent pegaptanib.

FIG. 15 is a graph depicting the results of 86 day in vivo release studyin rabbits dosed intravitreously with 5 mg of PLGA microparticlescontaining 15% weight percent pegaptanib compared to liquid pegaptanib.

FIG. 16 is a graph depicting the in vitro release profile of PLGApegaptanib microspheres suspended in PBS injection vehicle containing0.02% surfactant at a concentration of 100 mg microspheres permilliliter over an 8 month study.

FIG. 17 is a graph depicting the results of 8 month in vivo releasestudy in rabbits dosed intravitreously with 5 mg of PLGA microparticlescontaining 15% weight percent pegaptanib.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for thesustained delivery of a biologically active agent. In one aspect, thepresent invention provides compositions and methods for ocular sustaineddelivery of a biologically active agent. According to this aspect,syringable microsphere formulations are provided for administering abiologically active agent with a syringe via a 27-gauge needle orsmaller in order to provide sustained ocular levels of the biologicallyactive agent.

Microparticles can vary in size, ranging from submicron to millimeterdiameters. For ophthalmic applications, the diameters of themicroparticles range from about 1 micron (μm) to about 200 μm. Inanother embodiment, the microparticles have a particle size distributionranging from about 1 μm to about 100 μm in diameter. In anotherembodiment, the microparticles have a particle size distribution rangingfrom about 10 μm to about 50 μm in diameter.

In another embodiment, the microparticles have an average diameter ofless than about 45 μm. In another embodiment, the microparticles have anaverage diameter of about 30 μm. In another embodiment, themicroparticles have an average diameter of about 15 μm.

In one embodiment, the microparticles are of a suitable size andmorphology allowing for delivery though a needle having a smallinner-diameter such as a narrow (small) gauge needle. In a particularembodiment, the microparticles are of a suitable size and morphologyallowing for delivery though a needle suitable for ophthalmicadministration. Microparticles are referred to herein as “syringable” ifthe are able to be delivered though a needle. In one embodiment, themicroparticles are syringable through a medically acceptable needle. Inanother embodiment, the microparticles are syringable through a needlesuitable for ophthalmic administration. Example 7 provides methods foranalyzing suspendability and syringability.

In one particular embodiment particularly suited for ophthalmicapplications, the microparticles are syringable through a needle havinga gauge of at least 27 having a nominal inner diameter of 0.0075 inchesor less. In another particular embodiment the microparticles aresyringable through a needle having a gauge of at least 29 having anominal inner diameter of 0.0065 inches or less. In another particularembodiment the microparticles are syringable through a needle having agauge of at least 30 having a nominal inner diameter of 0.0055 inches orless.

In one embodiment, the microparticles have a mean diameter of less thanor equal to about 75% of the inner diameter of the needle used forinjection. In another embodiment, the microparticles have a meandiameter of less than or equal to about 50% of the inner diameter of theneedle used for injection. In another embodiment, the microparticleshave a mean diameter of less than or equal to about 25% of the innerdiameter of the needle used for injection. In another embodiment, themicroparticles have a mean diameter of less than or equal to about 10%of the inner diameter of the needle used for injection.

In one embodiment, the microparticles form free-flowing and/orun-agglomerated powders. Free-flowing and/or un-agglomerated powders areadvantageous because they roll with substantially no friction and can beeasily placed in containers, suspended or incorporated into a solutionsuitable for injection. Flowabilty of microparticles can be measured byany suitable means such as a Jenike Shear Tester (Jenike & Johanson,Inc., Westford, Mass.), which measures the direct shear strength ofpowders and other bulk solid materials. Using a Jenike Shear Tester, ashear cell (base and ring) is filled with material; a vertical load isapplied to the covered cell, using weights and the weight carrier; andthe shear cell ring is pushed horizontally across the base, with therequired force measured and recorded. Other apparatuses for measuringflowability include a powder rheometer (Freeman Technology,Worcestershire, UK) that measures the force of a twisted blade along ahelical path through a powder sample establishing a required flow rateand pattern of flow. A critical orifice and an angle of repose using anavalanche process may also be measured.

A chart representing particle size distribution of typicalmicroparticles before sieving is shown in FIG. 7.

The microparticles of the present invention have a core load sufficientto deliver the biologically active agent to maintain therapeuticallyeffective levels of the biologically active agent for sustained periods.In one embodiment, the microparticles have a core load of greater thanor equal to about 5% by weight of the biologically active agent. In oneembodiment, the microparticles have a core load of greater than or equalto about 10% by weight of the biologically active agent. In oneembodiment, the microparticles have a core load of greater than or equalto about 15% by weight of the biologically active agent. In anotherembodiment the microparticles have a core load of greater than or equalto about 20% by weight of the biologically active agent. In anotherembodiment the microparticles have a core load of greater than or equalto about 30% by weight of the biologically active agent.

In one embodiment, microparticles comprising pegaptanib have a core loadof greater than or equal to about 10% by weight of pegaptanib (2% byweight on an aptamer basis). In one embodiment, the microparticles havea core load of greater than or equal to about 15% by weight ofpegaptanib (about 3% by weight on an aptamer basis).

The microparticles of the present invention exhibit a low initial burst.In one embodiment, the microparticles exhibit an initial 24 hour invitro burst of less than or equal to about 10 wt % of the initial coreload of the biologically active agent. In one embodiment, themicroparticles exhibit an initial 24 hour in vitro burst of less than orequal to about 5 wt % of the initial core load of the biologicallyactive agent.

The microparticles of the present invention provide a sustained releaseof a biologically active agent. In one embodiment the microparticleshave an in vivo sustained release profile of at least 28 days (4 weeks/1month). In another embodiment the microparticles have an in vivosustained release profile of at least about 20, 40, 60 (2 months), 90 (3months), 180 days (6 months), or 365 days (12 months).

In one embodiment the microparticles encapsulate pegaptanib as thebiologically active agent and are adapted to release pegaptanib at arate ranging from about 0.01 to about 10 micrograms (μg) per day. Inanother embodiment, the microparticles encapsulate pegaptanib as thebiologically active agent and are adapted to release pegaptanib at arate ranging from about 0.1 to about 6 μg per day. In one particularembodiment pegaptanib microparticles release pegaptanib at a rate ofabout 0.025 μg, 0.25 μg, 0.5 μg, 0.75 μg, 1 μg, or 2 μg per day.

The microparticles of the present invention have a high encapsulationefficiency. In one embodiment, the microparticles have an encapsulationefficiency of greater than or equal to about 80%. In another embodimentthe microparticles have an encapsulation efficiency of greater than orequal to about 90%. In another embodiment the microparticles have anencapsulation efficiency of greater than or equal to about 95%. Inanother embodiment the microparticles have an encapsulation efficiencyof about 100%.

The microparticles of the present invention have any suitablemorphology. In one embodiment, the microparticles are solid. In anotherembodiment, the microparticles are smooth or non-pitted. In anotherembodiment, the microparticles are homogenous or monolithic. In anotherembodiment, the microparticles have a morphology allowing for a highcore load, high encapsulation efficiency, low burst, sustained releaseand syringability.

FIGS. 1 and 2 show images of examples of microparticles of the presentinvention. External morphological examination of the microparticles inFIG. 1 indicates that the microparticles are smooth and non-pitted.Internal morphological examination of the microparticles in FIG. 2indicates microparticles have a monolithic interior. Monolithicmicroparticles give consistent release kinetics.

Example 7 describes an experimental for the analysis of in vitro releaseof microparticle formulations of the present invention formed by theprocess as described in Examples 1 and 2. The results of the in vitrorelease analysis are depicted in FIGS. 9, 10, 13, and 16. The resultsshown in the figures demonstrate the sustained release properties of themicroparticles of the present invention. The results shown in thefigures also demonstrate that the microparticles of the presentinvention can be selectively designed to control the release of abiologically active agent over a desired time period.

In another aspect, the present invention provides methods ofadministering sustained release microparticle formulations to achieve adesired pharmacokinetic profile. According to one embodiment,microparticles comprising a biologically active agent are suspended in apharmaceutically acceptable solution and administered by syringe to theeye.

In another aspect, the present invention provides controlled release ofa biologically active agent in accordance with a desired pharmacokineticprofile. The microparticles of the present invention may be suspended ina solution containing an additional amount of the same biologicallyactive agent or a second biologically active agent. This solutioncontaining the dissolved biologically active agent may provide a desiredbolus of the agent to achieve a therapeutically effective level, whichis subsequently maintained for a prolonged period by the sustainedrelease microparticles.

According to one embodiment, microparticles comprising a biologicallyactive agent are suspended in a solution comprising the biologicallyactive agent and administered by syringe to the eye. In one embodiment,the microparticles are sustained release microparticles. In anotherembodiment, the microparticles are delayed release microparticles.Utilizing the vitreal residence time of the biologically active agent asa polymer clearance window allows the polymeric metabolites to becleared while sustaining a therapeutically relevant level within theeye. Utilizing the vitreal residence time of the biologically activeagent as a polymer clearance window also allows a staggered multiplebolus of a biologically active agent using a single administration. Sucha dosing regimen may be particularly useful for microencapsulatedbiologically active agents that have some residence time in thevitreous.

Examples of ocular formulations comprising a suspension of a particleincluding an ophthalmic drug and a liquid carrier containing at leastthe same ophthalmic drug is described in U.S. Pat. Nos. 4,882,150 and4,865,846, the contents of each are incorporated herein by reference intheir entirety.

In one embodiment, the regimen comprises the step of administering apharmaceutical formulation comprising a first bolus of a firstbiologically active agent and a delayed release microparticleformulation encapsulating a second bolus of the first biologicallyactive agent.

In a particular embodiment, the regimen comprises the step ofadministering about 100 μL of a pharmaceutical formulation comprising abolus of about 0.3 mg free pegaptanib in solution and delayed releasePLGA microparticles encapsulating about 35 mg pegaptanib. Themicroparticles have an initial burst of about 5-30% of pegaptanib andthen will release at a constant rate over about a 1-month time period.At the end of the microparticle release profile, a second burst willoccur releasing a second bolus of pegaptanib bringing the vitrealconcentration to about 0.3 mg. In another particular embodiment theregimen further comprises the step of administering a secondpharmaceutical formulation comprising pegaptanib at a time of four weekspost after the second burst, during which time the polymeric metabolitesare cleared. In this embodiment, the administration essentially gains anadditional month of efficacy.

One advantage of such a regimen includes the reduction of multipleintravitreous injections of polymer encapsulated biologically activeagents. Another advantage includes limiting any possible risk of havingthe clearance pathways of the eye hindered by a buildup of polymericbreakdown products causing detrimental effects on ocular function.

The microparticles of the present invention can be used for any purpose.In a particular embodiment, they are administered to a patient. They maybe administered to patients in a single or multiple dose. Themicroparticles may also be administered in a single dose form thatfunctions to release a biologically active agent over a prolonged periodof time, eliminating the need for multiple administrations.

In one aspect, the invention provides a method of treating or inhibitingan ocular disease state in a mammal in need thereof using any of themicrosphere compositions described herein. The method includesadministering the microparticles to a mammal in amounts sufficient totreat or inhibit the disease. In one embodiment, in order to treat anocular disorder, the microparticles are injected into the vitreous ofthe eye (intravitreous injection). In another embodiment, in order totreat an ocular disorder, the microparticles are disposed upon the outersurface of the sclera (sub-conjunctival injection). In such a system,once the biologically active agent is released out of the microparticle,the biologically active agent traverses the sclera to exert its effect,for example, reduce or inhibit the activity of a VEGF receptor, withinthe eye.

The microparticles may be used to treat a variety of ocular disordersincluding, for example, optic disc neovascularization, irisneovascularization, retinal neovascularization, choroidalneovascularization, corneal neovascularization, vitrealneovascularization, glaucoma, pannus, pterygium, macular edema, vascularretinopathy, retinal degeneration, uveitis, inflammatory diseases of theretina, and proliferative vitreoretinopathy. The cornealneovascularization to be treated or inhibited may be caused by trauma,chemical burns and corneal transplantation. The iris neovascularizationto be treated or inhibited may be associated with diabetic retinopathy,vein occlusion, ocular tumor and retinal detachment. The retinalneovascularization to be treated or inhibited may be associated withdiabetic retinopathy, vein occlusion, sickle cell retinopathy,retinopathy of prematurity, retinal detachment, ocular ischemia andtrauma. The intravitreous neovascularization to be treated or inhibitedmay be associated with diabetic retinopathy, vein occlusion, sickle cellretinopathy, retinopathy of prematurity, retinal s detachment, ocularischemia and trauma. The choroidal neovascularization to be treated or:inhibited may be associated with retinal or subretinal disorders, suchas, age-related macular degeneration, presumed ocular histoplasmosissyndrome, myopic degeneration, angioid streaks and ocular trauma.

Ophthalmic solutions are sterile solutions, essentially free fromforeign particles, suitably compounded and packaged for instillation orinjection into the eye. Preparation of an ophthalmic solution requirescareful consideration of such factors as the inherent toxicity of thedrug itself, isotonicity value, the need for buffering agents, the needfor a preservative (and, if needed, its selection), sterilization, andproper packaging.

While specific reference has been made to the use of the formulations ofthe present invention to administer biologically active agent to theeye, it is to be understood that the present invention can be used todeliver a biologically active agent to any desired site, including, butnot limited to, intraorbital, intraocular, intraaural, intratympanic,intrathecal, intracavitary, peritumoral, intratumoral, intraspinal,epidural, intracranial, and intracardial. While referring to the eye,the formulations of the present invention may be administeredintravitreously or periocularly (retrobulbarly, sub-tenonly,sub-conjunctivaly).

According to one embodiment, the microsphere formulations of the presentinvention are suitable for local application for the treatment ofvarious cancers. According to this embodiment, the microsphereformulations are injected locally at the tumor site or post-operativelyat the tumor site after tumor resection. According to this embodiment,microsphere formulations and related methods of use are provided fortreating various difficult to treat cancers such as glioblastomamultiforme, ovarian cancer, and head or neck cancer, for example.

A formulation of the invention may be used in the treatment of any eyedisease. A formulation of the invention may also be used to direct abiologically active agent to a particular eye tissue, e.g., the retinaor the choroid. The biologically active agent or combination of agentswill be chosen based on the disease, disorder, or condition beingtreated. In addition to a biologically active agent for a particularcondition, other compounds may be included for secondary effects, forexample, an antibiotic to prevent microbial growth. The amount andfrequency of the dosage will depend on the disease, disorder, orcondition being treated and the biologically active agent employed. Oneskilled in the art can make this determination.

By “treating” is meant the medical management of a patient with theintent that a cure, amelioration, stasis or prevention of a disease,pathological condition, or disorder will result. This term includesactive treatment, that is, treatment directed specifically towardimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the disease, pathological condition, or disorder. Inaddition, this term includes palliative treatment, that is, treatmentdesigned for the relief of symptoms rather than the curing of thedisease, pathological condition, or disorder; preventive treatment, thatis, treatment directed to prevention of the disease, pathologicalcondition, or disorder; and supportive treatment, that is, treatmentemployed to supplement another specific therapy directed toward theimprovement of the disease, pathological condition, or disorder. Theterm “treating” also includes symptomatic treatment, that is, treatmentdirected toward constitutional symptoms of the disease, pathologicalcondition, or disorder.

In one embodiment, the method of the invention provides a means forsuppressing or treating an ocular neovascular disorder. In someembodiments, ocular neovascular disorders amenable to treatment orsuppression by the method of the invention include ischemic retinopathy,iris neovascularization, intraocular neovascularization, age-relatedmacular degeneration, corneal neovascularization, retinalneovascularization, choroidal neovascularization, retinopathy ofprematurity, retinal vein occlusion, diabetic retinal ischemia, diabeticmacular edema, or proliferative diabetic retinopathy. In still anotherembodiment, the method of the invention provides a means for suppressingor treating psoriasis or rheumatoid arthritis in a patient in needthereof or a patient diagnosed with or at risk for developing such adisorder.

As used herein, the terms “neovascularization” and “angiogenesis” areused interchangeably. Neovascularization and angiogenesis refer to thegeneration of new blood vessels into cells, tissue, or organs. Thecontrol of angiogenesis is typically altered in certain disease statesand, in many cases, the pathological damage associated with the diseaseis related to altered, unregulated, or uncontrolled angiogenesis.Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, including those characterized by the abnormal growth byendothelial cells, and supports the pathological damage seen in theseconditions including leakage and permeability of blood vessels.

By “ocular neovascular disorder” is meant a disorder characterized byaltered or unregulated angiogenesis in the eye of a patient. Exemplaryocular neovascular disorders include optic disc neovascularization, irisneovascularization, retinal neovascularization, choroidalneovascularization, corneal neovascularization, vitrealneovascularization, glaucoma, pannus, pterygium, macular edema, diabeticretinopathy, diabetic macular edema, vascular retinopathy, retinaldegeneration, uveitis, inflammatory diseases of the retina, andproliferative vitreoretinopathy.

In addition to treating pre-existing neovascular disorders, the therapythat includes a biologically active agent can be administeredprophylactically in order to prevent or slow the onset of thesedisorders. In prophylactic applications, the biologically active agentis administered to a patient susceptible to or otherwise at risk of aparticular neovascular disorder. The precise timing of theadministration and amounts that are administered depend on variousfactors such as the patient's state of health, weight, etc.

A “biologically active agent”, “biologically active moiety” or“biologically active molecule” can be any substance which can affect anyphysical or biochemical properties of a biological organism, includingbut not limited to, viruses, bacteria, fungi, plants, animals, andhumans. Biologically active molecules can include any substance intendedfor diagnosis, cure mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals.

Examples of biologically active agents include, but are not limited to,nucleic acids, nucleosides, oligonucleotides, antisenseoligonucleotides, RNA, DNA, siRNA, RNAi, aptamers, antibodies, peptides,proteins, enzymes, fusion proteins, porphyrins, and small moleculedrugs. Other biologically active agents include, but are not limited to,dyes, lipids, cells, viruses, liposomes, microparticles and micelles.Examples of antibodies include, but are not limited to, anti-VEGFantibodies bevacizumab (Avastin™) and ranizumab (Lucentis™). Examples ofaptamers include, but are not limited to, pegaptanib (Macugen®).Examples of porphyrins include, but are not limited to, verteporfin(Visudine®). Examples of steroids include, but are not limited to,anecortave acetate (Retaane®). Examples of fusion proteins include, butare not limited to, VEGF Trap™ (Regeneron Pharmaceuticals, Inc.Tarrytown, N.Y.). Examples of RNAi include, but are not limited to,Direct RNAi™ (Alnylam Pharmaceuticals, Cambridge, Mass.).

Classes of biologically active agents that are suitable for use with theinvention include, but are not limited to, antibiotics, fungicides,anti-viral agents, anti-infective agents, anti-inflammatory agents,anti-tumor agents, anti-tubulin agents, cardiovascular agents,anti-anxiety agents, hormones, growth factors, steroidal agents, and thelike.

The term “anti-VEGF agent” refers to any biologically active agent wherethe primary mode of action is to (a) impair binding of any VEGF isoformto its receptor, or (b) block signaling of VEGF receptors R1 and R2.

It will be understood that pharmaceutically acceptable salts of thebiologically active molecule disclosed herein are also included in thepresent invention and can be used in the compositions and methodsdisclosed herein.

The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars and inter-sugar (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly.Incorporation of substituted oligomers is based on factors includingenhanced cellular uptake, or increased nuclease resistance and arechosen as is known in the art. The entire oligonucleotide or onlyportions thereof may contain the substituted oligomers.

As used herein, the term “aptamer” means any polynucleotide, or saltthereof, having selective binding affinity for a non-polynucleotidemolecule via non-covalent physical interactions. An aptamer can be apolynucleotide that binds to a ligand in a manner analogous to thebinding of an antibody to its epitope. The target molecule can be anymolecule of interest. An example of a non-polynucleotide molecule is aprotein. An aptamer can be used to bind to a ligand-binding domain of aprotein, thereby preventing interaction of the naturally occurringligand with the protein.

“Anti-VEGF aptamers” or “VEGF aptamers” are meant to encompasspolynucleotide aptamers that bind to, and inhibit the activity of, VEGF.Such anti-VEGF aptamers may be RNA aptamers, DNA aptamers or aptamershaving a mixed (i.e., both RNA and DNA) composition. Such aptamers canbe identified using known methods. For example, Systematic Evolution ofLigands by Exponential enrichment, or SELEX, methods can be used asdescribed in U.S. Pat. Nos. 5,475,096 and 5,270,163, each of which areincorporated herein by reference in its entirety. Anti-VEGF aptamersinclude the sequences described in U.S. Pat. Nos. 6,168,778, 6,051,698,5,859,228, and 6,426,335, each of which are incorporated herein byreference in its entirety. The sequences can be modified to include5′-5′ and/or 3′-3′ inverted caps. (See Adamis, A. P. et al., publishedapplication No. WO 2005/014814, which is hereby incorporated byreference in its entirety).

For ophthalmic drug delivery applications, exemplary disease statesinclude macular degeneration (dry and wet), diabetic retinopathy,glaucoma, optic disc neovascularization, iris neovascularization,retinal neovascularization, choroidal neovascularization, pannus,pterygium, macular edema, vascular retinopathy, retinal vein occlusion,histoplasmosis, ischemic retinal disease, retinal degeneration, uveitis,inflammatory diseases of the retina, keratitis, cytomegalovirusretinitis, an infection, conjunctivitis, cystoid macular edema, cancer,and proliferative vitreoretinopathy.

Classes of biologically active agents include anti-infectives including,without limitation, antibiotics, antivirals, and antifungals;analgesics; antiallergenic agents; mast cell stabilizers; steroidal andnon-steroidal anti-inflammatory agents; decongestants; anti-glaucomaagents including, without limitation, adrenergics, beta-adrenergicblocking agents, alpha-adrenergic blocking agonists, parasympathomimeticagents, cholinesterase inhibitors, carbonic anhydrase inhibitors, andprotaglandins; antioxidants; nutritional supplements; angiogenesisinhibitors; antimetabolites; fibrinolytics; wound modulating agents;neuroprotective drugs; angiostatic steroids; mydriatics; cyclopegicmydriatics; miotics; vasoconstrictors; vasodilators; anticlottingagents; anticancer agents; immunomodulatory agents; VEGF antagonists;immunosuppresant agents; and combinations and prodrugs thereof.

The biologically active agent may be conjugated to non-toxic long-chainpolymers such as poly(ethylene glycol) (PEG). Such polymers may increaseblood circulation lifetimes, improve efficacy and safety, and increasestability. Competitive binding studies of aptamers utilizing thisstrategy revealed that the PEG unit actively assists in the inhibitorypotency of Macugen, specifically with its ability to prevent VEGFbinding to the Flt-1 receptor. (US Patent Application Publication No. US2005/0260651, which is hereby incorporated herein by reference in itsentirety) While the clinical relevance is uncertain, strong evidenceindicates that inhibition of Flt-1 binding is a major contributor to theanti-inflammatory properties of Macugen (Usui, T. et al. (2004)“VEGF164(165) as the Pathological Isoform: Differential Leukocyte andEndothelial Responses through VEGFR1 and VEGFR2.” I.O.V.S. 45(2), 368).

Specific biologically active agents include MACUGEN® (pegaptanib sodiuminjection) as described in U.S. Pat. No. 6,051,698, herein incorporatedin its entirety by reference. Pegaptanib is also referred to as EYE001(previously referred to as NX1838).

Pegaptanib is a covalent conjugate of an oligonucleotide of twenty-eightnucleotides in length that terminates in a pentylamino linker, to whichtwo 20-kilodalton (kDa) monomethoxypolyethylene glycol (PEG) units arecovalently attached via the two amino groups on a lysine residue.Pegaptanib is optionally provided in the form of a pharmaceuticallyacceptable salt. In one embodiment, pegaptanib is provided as pegaptanibsodium. The molecular formula for pegaptanib sodium isC₂₉₄H₃₄₂F₁₃N₁₀₇Na₂₈O₁₈₈P₂₈(C₂H₄O)_(n) (where n is approximately 900) andthe molecular weight is approximately 50 kDa.

The chemical name for pegaptanib sodium is as follows: RNA,((2′-deoxy-2′-fluoro)C-G_(m)-G_(m)-AA-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)C-A_(m)-G_(m)-(2′-deoxy-2′-fluoro)U-G_(m)-A_(m)-A_(m)-(2′-deoxy-2′-fluoro)U-G_(m)-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)U-A_(m)-(2′-deoxy-2′-fluoro)U-A_(m)-(2′-deoxy-2′-fluoro)C-A_(m)-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)C-G_(m)-(3′→3′)-dT),5′-ester withα,α′-[4,12-dioxo-6-[[[5-(phosphoonoxy)pentyl]amino]carbonyl]-3,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[(ω-methoxypoly(oxy-1,2-ethanediyl)],sodium salt.

Dosage levels of pegaptanib sodium on the order of about 1 μg/kg to 100mg/kg of body weight per administration are useful in the treatment ofneovascular disorders. Examples of formulations are found in WO03/039404, which is hereby incorporated by reference in its entirety. Insome embodiments, pegaptanib sodium is administered at a dosage of about0.1 mg to about 1.0 mg locally into the eye, wherein the treatment iseffective to treat occult, minimally classic, and predominantly classicforms of wet macular degeneration. When administered directly to theeye, the dosage range is about 0.3 mg to about 3 mg per eye, in someembodiments the dosage range is about 0.1 mg to about 1.0 mg per eye. Inone embodiment, pegaptanib sodium is administered in a therapeuticallyeffective amount of about 0.1-3.0 mg, 0.1-1.0 mg, or about 0.3 mg. Inone embodiment, pegaptanib sodium is present in an ophthalmic injectionsolution formulation at a concentration ranging from 0.1 to 3.0 mg/mL.According to one embodiment, the carrier comprises sodium phosphate andsodium chloride. According to one specific embodiment the carriercomprises 10 mM sodium phosphate and 0.9% sodium chloride.

According to one embodiment, the dose is effective to achieve a vitreousconcentration of the anti-VEGF aptamer of about 10-30 ng/mL. Accordingto another embodiment, the dose is effective to maintain a vitreousconcentration of the anti-VEGF aptamer of about 10-30 ng/mL throughoutthe administration period.

In alternative embodiments, the anti-VEGF agent is an anti-VEGF aptamerand is administered at a dosage of less than 0.3 mg to about 0.003 mglocally into the eye. In some embodiments, the anti-VEGF aptamer isadministered at a dosage less than about 0.30 mg. Examples of suchformulations are found in U.S. Patent Application Ser. No. 60/692,727;which is incorporated herein by reference in its entirety.

As used herein, “microparticles” refers to particles having a diameterof typically less than 1.0 mm, and more typically between 1.0 and 250microns.

The microparticles of the present invention include, but are not limitedto, microspheres, microcapsules, microsponges, microgranules andparticles in general, with an internal structure comprising a matrix ofagent and excipient. Microparticles may also include nanoparticles.

Microspheres are typically solid spherical microparticles. Microcapsulesare typically spherical microparticles typically having a core of adifferent polymer, drug, or composition.

As used herein, the term “nanoparticles” refers to particles having adiameter of typically between about 20 nanometers (nm) and about 2.0microns (μm), typically between about 100 nm and about 1.0 μm.

An “injection” is a preparation intended for parenteral administration.Injections include, but are not limited to, liquid preparations that aredrug substances or solutions or suspensions thereof.

The grammatically correct and preferred term “intravitreous” is usedherein and in the art. The term “intravitreal” is used colloquially asan alternative to the term “intravitreous” for injections into the eye'svitreous humor between the lens and the retina.

The term “controlled release” refers to control of the rate and/orquantity of biologically active molecules delivered according to thedrug delivery formulations of the invention. The controlled releasekinetics can be continuous, discontinuous, variable, linear ornon-linear. This can be accomplished using one or more types of polymercompositions, drug loadings, inclusion of excipients or degradationenhancers, or other modifiers, administered alone, in combination orsequentially to produce the desired effect. “Controlled release”microparticles include, but are not limited to, “sustained release”microparticles and “delayed release” microparticles.

The term “sustained release” refers to releasing a biologically activeagent into the body steadily, over an extended period of time. Sustainedrelease formulations offer the ability to provide a subject with abiologically active agent over a time period greater than that achievedby a typical bolus administration of the biologically active agent.Sustained release microparticles may advantageously reduce the dosingfrequency of a biologically active agent.

“Zero-order” or “linear release” is generally construed to mean that theamount of the biologically active molecule released over time remainsrelatively constant as a function of amount/unit time during the desiredtime frame. Multi-phasic is generally construed to mean that releaseoccurs in more than one “burst”.

The term “packed bed apparatus” refers to any vessel containing packingmaterial capable of creating an emulsion upon contact with twoimmiscible fluids.

The term “biodegradable” or “bioerodible,” as used herein, refer topolymers that dissolve or degrade within a period that is acceptable inthe desired application (usually in vivo therapy), typically less thanabout five years, and more preferably less than about one year, onceexposed to a physiological solution of pH ranging from about 6 to about9 and at a temperature of ranging from about 25 C to about 38 C.

A variety of biodegradable polymers used for controlled releaseformulations are well known in the art. Suitable polymers for exampleinclude, but are not limited to, poly(hydroxy acids), poly(lactic acid),poly(glycolic acid), poly(lactic acid-co-glycolic acid),polycaprolactones, polyanhydrides, polycarbonates, polyamides,polyesters, polyorthoesters, polyhydroxybutryate, certain types ofprotein and polysaccharide polymers, and blends, copolymers or mixturesthereof.

The biodegradable polymers are optionally capped or un-capped. Cappedpolymers include, but are not limited to, those having esterified oramidated end groups. Un-capped polymers include free hydroxyl orcarboxyl end-groups. In one embodiment, the microparticles comprisefree-acid poly (lactic acid-co-glycolic acid). In another embodiment,the microparticles comprises lauryl or N-capped poly(lacticacid-co-glycolic acid).

Preferred polymers include poly (hydroxy acids). In one embodiment thepolymer is poly (lactic acid-co-glycolic acid) (“PLGA”) that degrade byhydrolysis following exposure to the aqueous environment of the body.The polymer is then hydrolyzed to yield lactic and glycolic acidmonomers, which are normal byproducts of cellular metabolism. The rateof polymer disintegration can vary from several weeks to periods ofgreater than one year, depending on several factors including polymermolecular weight, ratio of lactide to glycolide monomers in the polymerchain, and stereoregularity of the monomer subunits (mixtures of L and Dstereoisomers disrupt the polymer crystallinity enhancing polymerbreakdown). Microparticles may contain blends of two and morebiodegradable polymers, of different molecular weight and/or monomerratio.

PLGA may have any suitable monomer ratio of lactide:glycolide. In oneembodiment the amount of lactide ranges from 40-100%. In anotherembodiment the amount of glycolide ranges from 0-60%. In one embodiment,the PLGA has a monomer ratio of lactide:glycolide in the range of about40:60 to 100:0. In another embodiment, the PLGA has a monomer ratio oflactide:glycolide in the range from about 45:55 to 100:0. In oneparticular embodiment, the PLGA has a monomer ratio of lactide:glycolideof about 50:50. In another particular embodiment, the PLGA has a monomerratio of lactide:glycolide of about 65:35. In another particularembodiment, the PLGA has a monomer ratio of lactide:glycolide of about75:25. The particular ratio of the polymers may be determined based onpharmacokinetic evaluations.

The microparticles may release a biologically active agent by anysuitable means to allow for a controlled release of the biologicallyactive agent. While not wishing to be bound by theory, themicroparticles can release the biologically active agent by bulkerosion, diffusion or a combination of both.

A surfactant is optionally used in order to provide formulations thathave the required syringability. In one embodiment, a surfactant is usedfor providing a stable emulsion during the process of forming themicroparticles of the present invention. In another embodiment, asurfactant is used for preventing agglomeration during lyophilizationduring the process of forming the microparticles. In another embodiment,a surfactant is used for preventing agglomeration within the injectionvehicle during the process of delivering the microparticles. Withoutwishing to be bound by theory, surfactants may provide batch-to-batchconsistency of microparticles by forming a thin layer of material aroundthe microparticles that helps prevent clumping. Any suitable surfactantmay be used. Suitable surfactants include, but are not limited to,cationic, anionic, and nonionic compounds such as poly(vinyl alcohol),carboxymethyl cellulose, lecithin, gelatin, poly(vinyl pyrrolidone),polyoxyethylenesorbitan fatty acid ester (Tween 80, Tween 60, Tween 20),sodium dodecyl sulfate (SDS), mannitol and the like.

In one embodiment, the microparticles are formed using an emulsioncomprising poly (vinyl alcohol). In a particular embodiment, themicroparticles are formed using an emulsion comprising 1.0% poly (vinylalcohol). The concentration of surfactant in the process medium isestablished to be an amount sufficient to stabilize the emulsion.

In one embodiment, the microparticles are lyophilized in a solutioncomprising SDS, Tween 20 or mannitol. In one particular embodiment, themicroparticles are lyophilized in a solution comprising 7.8% SDS.

In one embodiment, the microparticles are suspended in an injectionsolution comprising SDS, Tween 20 or mannitol. In one particularembodiment, the microparticles are suspended in an injection solutioncomprising 0.5% SDS.

In one embodiment, the inherent viscosity of the biodegradable polymermay be in the range 0.1 to 2.0 dL/g. In another embodiment, the inherentviscosity of the biodegradable polymer ranges from about 0.1 to about1.0 dL/g. In another embodiment, the inherent viscosity of thebiodegradable polymer is about 0.16 dL/g, 0.35 dL/g or 0.61 dL/g.

Derivatized biodegradable polymers are also suitable for use in thepresent invention, including hydrophilic polymers attached to PLGA andthe like. To form microparticles, in particular, a variety of techniquesknown in the art can be used. These include, for example, single ordouble emulsion steps followed by solvent removal. Solvent removal maybe accomplished by extraction, evaporation or spray drying among othermethods.

In a typical solvent extraction method, a polymer is dissolved in acontinuous phase (e.g., an organic solvent) that is at least partiallysoluble in a discontinuous phase (e.g., an extraction solvent such aswater). A biologically active molecule, either in soluble form ordispersed as fine particles, is then added to the polymer solution, andthe mixture is dispersed into an aqueous phase that contains asurface-active agent such as poly (vinyl alcohol). The resultingemulsion is added to a larger volume of water where the organic solventis removed by extraction or evaporation from the polymer/biologicallyactive agent to form hardened microparticles.

Microparticles of the present invention may be prepared using anysuitable method. In one embodiment, the microparticles are preparedusing an emulsion process. A general scheme illustrating the process isshown in FIG. 8. In general, the methods combine a first phase and asecond phase and pass the combination though an emulsifier. After anemulsion is formed, the solvent of the first phase is removed from theemulsion in an extractor, producing hardened microparticles. Themicroparticles are then sieved and dried.

In one embodiment, the methods have a first phase consisting of anorganic solvent, a polymer and a biological or chemical agent dissolvedor dispersed in the first solvent. The second phase comprises water anda stabilizer and, optionally, the first solvent. The first and thesecond phases are emulsified and, after an emulsion is formed, the firstsolvent is removed from the emulsion, producing hardened microparticles.

In another embodiment, the methods use a non-aqueous oil-in oil (o/o)process for making microparticles. In general an o/o method includes thesteps of: a) dissolving a biocompatible polymer in a solvent to form asolution; b) combining a biologically active agent with the solution toproduce a mixture; c) optionally combining the mixture of step (b) witha coacervating agent (optionally, while homogenizing the solution); andd) permitting the biocompatible polymer to form microparticlescontaining the biologically active agent. (See PCT Publication No. WO03/092665 and K. G. Carrasquillo et al., “Controlled Delivery of theAnti-VEGF Aptamer EYE001 with Poly(lactic-co-glycolic) AcidMicrospheres,” I.O.V.S. (2003) 44(1), 290, each of which are herebyincorporated herein by reference in their entirety.)

In another embodiment, the methods use a water-in-oil-in-water (w/o/w)process for making microparticles. Such microparticles can be preparedby forming a primary water-in-oil emulsion comprising a water solublemolecule-containing solution as the inner aqueous phase and apolymer-containing solution as the oil phase. This primary emulsion canthen be dispersed in an outer aqueous phase to form the finalwater-in-oil-in-water emulsion. The w/o/w tri-phasic emulsion can thenbe allowed to stir for a set period of time to promote extraction andevaporation of the organic solvent in which the solvent of the oil phaseis removed and results in formation of hardened microparticles. Examplesof w/o/w emulsion processes are described in U.S. Pat. Nos. 4,954,298;5,330,767; 5,851,451 and 5,902,834, each of which are herebyincorporated herein by reference in their entirety. FIG. 5 illustratesan exemplary w/o/w process for forming microparticles of the presentinvention. Microparticles of the present invention, formed by a w/o/wprocess, are described in Example 2.

Emulsions may be formed by any suitable method. In one embodiment, abatch device for mixing the first and second phases under turbulentconditions such as with a stirrer as disclosed in U.S. Pat. No.5,407,609, which is hereby incorporated herein by reference in itsentirety. Other batch processes may employ a homogenizer or a sonicator.In another embodiment, an emulsion is formed by continuously mixing thefirst phase and second phase, in-line, using turbulent flow conditions,as in the use of an in-line dynamic mixer or an in-line static mixersuch as described in U.S. Pat. No. 5,654,008, which is herebyincorporated herein by reference in its entirety.

In one embodiment, the microparticles are prepared according to theprocess disclosed in PCT publication No. WO 2005/003180, which is herebyincorporated by reference in its entirety, which discloses anemulsion-based technique employing a packed bed system that uses laminarflow conditions to produce an emulsion that results in microparticlescontaining biological or chemical agents after solvent removal.

The packed bed apparatus for the production of microparticles through anemulsion-based technique may be a vessel of any shape capable of beingfilled with packing material that allows liquid to flow through it (SeeFIG. 6). The apparatus of the present invention may further provide amaterial capable of insertion into both ends for enclosure of materialsin such apparatus. FIG. 6 illustrates an exemplary apparatus accordingto one embodiment of the present invention. In this embodiment, a tube(1) is filled with beads as packing material (2).

Microparticle morphology is observed by Scanning Electron Microscopy(SEM) analysis. Microparticles are sputter coated with gold using anAnatech LTD Hummer 6.2 system. Scanning electron microscopic images weretaken using a JEOL JSM-5600 scanning electron microscope andaccompanying software at an accelerating voltage of 5-10 keV. Forselected samples, SEM analysis of the internal microsphere structure wasmade after embedding microparticles in L.R. White Resin and thensplitting the preparation after the resin hardened. Sample images areshown in FIGS. 1 and 2.

The microparticles of the present invention can be stored as a drymaterial such as a sterile lyophilized (or freeze dried) powder. In theinstance of administration to a patient, prior to such use, the drymicroparticles can be suspended in any pharmaceutically acceptablevehicle. Upon suspension, the microparticles may then be injected intothe patient or otherwise utilized. Suitable pharmaceutically acceptablevehicles include, but are not limited to, a liquid vehicle, a suspensionvehicle or an injection vehicle. The vehicle may include a surfactant,such as SDS, Tween 20 or mannitol.

Microparticles may be present in any suitable formulation. Methods wellknown in the art for making formulations are found, for example, inRemington: The Science and Practice of Pharmacy (20th ed., A. R. Gennaroed., Lippincott: Philadelphia, 2000). Microparticles may be administeredto humans, domestic pets, livestock, or other animals with apharmaceutically acceptable diluent, carrier, or excipient. In oneembodiment, the microparticles may be present in any suitableformulation for delivery to the eye.

Conventional pharmaceutical practice may be employed to provide suitableformulations or compositions to administer the identified compound topatients suffering from a disease, disorder, or condition of the eye.Administration may begin before, during, or after the patient issymptomatic.

In one embodiment, the microparticles are suspended in an acceptablepharmaceutical liquid vehicle, such as a 2.5 wt. % solution ofcarboxymethyl cellulose in water. In another embodiment, pegaptanibmicroparticles are suspended in an aqueous solution comprising 10 mMSodium Phosphate, 136.9 mM Sodium Chloride, 2.7 mM Potassium Chloride,0.05% Tween 20, and pH 7.4 (Filtered 0.2 μm). In another embodiment,pegaptanib microparticles are formulated for a dose of 1 mg ofpegaptanib per 100 μL of microparticle solution. In another embodiment,pegaptanib microparticles are suspended in a solution comprisingpegaptanib. In a particular embodiment, pegaptanib microparticles aresuspended in a solution comprising pegaptanib formulated at 3.47 mg/mL,measured as the free acid form of the oligonucleotide, sodium chloride,monobasic sodium phosphate monohydrate, dibasic sodium phosphateheptahydrate, hydrochloric acid, and/or sodium hydroxide to adjust thepH and water for injection.

The volume of injection will depend on the route of administration. Atypical intravitreous injection requires a 27 gauge needle or narrowerand a delivered volume less than or equal to about 150 μL. A typicalsubconjunctival injection can accommodate a 23 gauge needle and a volumeof up to 750 μL.

Sustained release formulations can be advantageous by reducingintravitreous (IVT) dosing frequencies of therapy involving biologicallyactive agents. Applicants evaluated the in vivo release properties ofpegaptanib-loaded microparticles as set forth in Example 9 and therebydemonstrated the feasibility of delivering pegaptanib over a period ofapproximately one month or more from a poly(lactide-co-glycolide) (PLGA)polymer based sustained release formulation.

Elimination of pegaptanib from the eye into systemic circulation isconsidered to be the rate-limiting step of pegaptanib's plasmapharmacokinetics. Therefore the plasma pharmacokinetics of pegaptanibshould mirror the in vivo release of pegaptanib from the sustainedrelease formulation. Following bilateral intravitreous (IVT)administration of liquid pegaptanib (pegaptanib sodium in phosphatebuffered saline solution), plasma concentrations declined slowlyovertime. The terminal phase rate constant, in plasma, reflected theslow absorption of pegaptanib from the eye into the systemic circulationafter an IVT injection (see FIG. 12). Since it is expected thatpegaptanib will be released in a sustained fashion from PLGAformulations into the vitreous, it is expected that the pegaptanibreleased into the vitreous will have distribution and clearanceproperties similar to those of pegaptanib administered by a phosphatebuffered saline solution.

Plasma concentrations resulting from a 28 day in vivo oculardistribution study in rabbits dosed intravitreously with 5 mg of PLGAmicroparticles containing 15% weight percent pegaptanib are shown inFIG. 12. The results show that the microparticles have a low burstrelease. A large burst release, common to PLGA formulations containingwater soluble or hydrophilic compounds, is absent. In addition, plasmapegaptanib concentration levels were measured at a relatively constantlevel between 0.05-0.4 nM over the 28 day study period relative to anequivalent IVT liquid pegaptanib dose, indicating a sustained release ofpegaptanib in the vitreous was achieved.

It is known in the art that modifying the polymer composition of asustained release microparticle formulation affects the rate of polymerdecomposition in vivo and therefore effects the release characteristicsof the microparticle formulation. Therefore demonstrating a releaseduration of one month from a microparticle formulation in vivo indicatesthat a release duration of greater than one month from a microparticleformulation in vivo is feasible.

EXAMPLES

The following examples serve to illustrate certain useful embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof. Alternative materials and methods can beutilized to obtain similar results.

Example 1 Preparation of Microparticles (Oil-in-Water)

Formulations were prepared via an oil-in-water solventextraction/evaporation method. Macugen®; pegaptanib sodium ((OSI)Eyetech, Inc., NY, N.Y.) and PLGA were dissolved in methylene chlorideand an emulsion was formed according to the process disclosed in PCTpublication No. WO 2005/003180, which is incorporated herein byreference in its entirety. Following solvent extraction from theemulsion particles, the hardened microparticles were sieved through a 45μm screen. Microparticles≦45 μm were collected by centrifugation anddried by lyophilization.

Example 2 Preparation of Pegaptanib Microparticles(Water-in-Oil-in-Water)

A batch size of 200 milligrams dry microspheres containing pegaptanibwas prepared according to the following procedure:

Step 1. Preparation of Primary Aqueous Phase

-   -   a. 30 mg Pegaptanib    -   b. 300 μL water    -   c. The mixture was vortexed to dissolve components        Step 2. Preparation of Organic Phase    -   a. 200 mg PLGA (i.e. 50:50 lactide:glycolide, IV=0.37 dL/g)    -   b. 7 ml methylene chloride (CH₂Cl₂)    -   c. The mixture was vortexed to dissolve components        Step 3. Preparation of Secondary Aqueous Phase/Quench Solution    -   b. 10.2 g polyvinyl alcohol    -   c. 104 g sucrose    -   d. 1.25 mL 1M Tris, pH 8.0    -   e. 1 mL 0.5M EDTA, pH 8.0    -   f. All components were dissolved in ˜800 mL water. The pH was        adjusted to 7.4 and the final volume to was brought to 1 L.        Step 4.

The organic solution was homogenized at 20000 RPM for a total of 2minutes using a Virtis homogenizer. While homogenizing, the primaryaqueous (drug containing) solution was slowly injected through 21Gneedle over 20 seconds to form primary water-oil emulsion.

Step 5.

The secondary aqueous phase (35 mL) was homogenized at 20000 RPM for 1minute. The primary emulsion was immediately transferred into a beakercontaining homogenized secondary aqueous phase to form a secondarywater-oil-water emulsion.

Step 6.

The secondary emulsion was poured into a 250 mL beaker containing 100 mLquench solution (stirring speed=4) to extract CH₂Cl₂. Allowed to stir atroom temperature for 3.5 hours.

Step 7.

The material was transferred to a collection vessel for centrifugationand washing.

-   -   a. Centrifuged at 1500 RPM for 15 minutes and pour off        supernatant.    -   b. Repeated centrifugation and decanting of supernatant two more        times.        Step 8.

The final pellet was re-suspended in DI water and lyophilize for about96 hours (4 days). During first 24 hours on lyophilizer, the vacuum onsystem was purged every 2 hours.

Example 3 Morphology Analysis of Microparticles

Microparticle morphology was observed by Scanning Electron Microscopy(SEM) analysis. Microparticles were sputter coated with gold using anAnatech LTD Hummer 6.2 system. Scanning electron microscopic images weretaken using a JEOL JSM-5600 scanning electron microscope andaccompanying software at an accelerating voltage of 5-10 keV. Forselected samples, SEM analysis of the internal microsphere structure wasmade after embedding microparticles in L.R. White Resin and thensplitting the preparation after the resin hardened.

Scanning electron micrograph images of microparticles formed by theprocess as set forth in Example 1 are shown in FIGS. 1 and 2. The imagesof FIG. 1 show that the microparticles have a smooth externalmorphology. The image of FIG. 2 shows that the microparticles have amonolithic internal morphology.

A scanning electron micrograph image of microparticles formed by thewater-in-oil-in water process as set forth in Example 2 are shown inFIG. 3. The image shows that the microparticles have a particle size ofless than about 10 μm and a smooth external morphology.

The in vitro and in vivo release analysis methods as set forth inExample 7 and Example 8 below indicate that the smooth, monolithicmorphology of the microparticles release pegaptanib in a sustainedrelease manner with a low burst.

Example 4 Analysis of Pegaptanib Coreload and Purity

Pegaptanib microspheres were prepared as set forth in Example 1 above.The microspheres were analyzed to determine if pegaptanib was degradedduring their preparation. The microsphere preparations werecharacterized for Pegaptanib content and purity by HPLC. Approximately6.5 mg of formulation was accurately weighed into a 2 mL microcentrifugetube. The formulation was dissolved in 1.0 mL of 5% (water)/95%(acetonitrile), and the polymer precipitated by the addition of 1.0 mL10-mM Na-phosphate pH 2.5. The resulting cloudy suspension was vortexedand clarified by centrifugation at 14,000 rpm for 2 minutes. Clearsupernatant was assayed by HPLC. Samples were assayed against a 670.8μg/mL EYE-001 (equivalent to 126.5 μg/mL oligomer) standard solutionprepared in water and stored at −80° C.

FIG. 8 shows a RP-HPLC chromatogram used to measure the core load andpurity of pegaptanib extracted from the microspheres. The chromatogramshows a high core load (12.3%) and that the purity of pegaptanib(99.18%) is unchanged by the formulation process.

Example 5 Analysis of Surfactant Effect on Lyophilization

Microparticles were prepared with 50:50-2.5 A (Alkermes) polymer and a15% initial load of pegaptanib as set forth in Example 1. Followingsolvent extraction the microspheres were sieved through a 45 μm screenand collected on a 25 μm screen. The microspheres were washed with waterand the batch was split in three. One fraction was lyophilized in water.The second fraction was lyophilized in SDS, such that the dried productwas 7.8% (w/w) SDS. The third fraction was lyophilized in Tween20, suchthat the dried product was 0.24% (w/w) Tween20. In order to gaugesuspendability and syringability, 100 μl vehicle was added to 20 mgpegaptanib microspheres in a 1.5 ml centrifuge tube. The followingvehicles were tested: PBS; PBS with 0.5% SDS; PBS with 0.02% Tween20;PBS with 0.5% CMC; and PBS with 0.5% CMC and 0.2% Tween20. The mixturewas suspended by tapping to determine ease of suspendability. Themixture was then vortexed for several seconds: The mixture was examinedfor uniformity of suspension and syringability. The results are shown inTable 1.

TABLE 1 077-141 077-141 077-151 077-141 0.24% Tween20 7.8% SDS 2%Mannitol PBS Suspend easily Suspend OK Visibly Clumpy Inject OK, slightSome sticks to side Sticks to side clogging Inject OK, slight Injects OKat first, Little force required clogging worst with time after settlingLittle force required after settling PBS + 0.02% Difficult to Suspendeasily Tween 20 suspend Injects well Inject Fine Little force requiredSettle quickly after settling PBS + 0.5% SDS Suspend easily Injects wellLittle force required after settling Foamy PBS + 0.5% CMC Suspend OKSuspend OK Injects with some Inject OK clogging Even after settlingPBS + 0.5% CMC + Very difficult to Suspend easily Injects OK 0.02% Tween20 suspend Injects well Even with settling Clog initially then Stays insuspension OK well

Example 6 Analysis of Suspendability and Syringability

Aliquots of 10, 20 or 30 mg of pegaptanib microspheres, prepared by themethod as set forth in Example 1, were added to 100 μL vehicle (0.5% CMCand 0.05% SDS in PBS). The mixture was examined for uniformity ofsuspension. The solution was then drawn into a 1 cc syringe through a ½inch, 27 gauge needle and held for a time before pushing the sample outthrough the same needle. The procedure was repeated for a ½ inch, 29gauge needle. The test was repeated after allowing microspheres tosettle toward the syringe hub for 30 seconds. The following vehicleswere tested: Saline; PBS; PBS with 0.05 or 0.5% SDS; PBS with 0.02%Tween20; PBS with 1% Mannitol; PBS with 0.5% SDS and 0.5% CMC; and PBSwith 0.02% Tween20 and 0.5% CMC. The results are shown in Tables 2 and3.

TABLE 2 100 mg 200 mg microspheres/ml 150 mg/ml microspheres/ml SalineVisibly clumpy Sticks to tube Injects fine PBS Sticks to tube Injectsfine PBS + 0.1% Sticks to tube SDS Injects fine Force required aftersettling PBS + 0.5% Injects fine Difficult to SDS Injects fine aftersuspend Settles settling Foamy quickly Clogs PBS + 0.02% Cloggedinitially, then Difficult to Difficult to Tween₂₀ injected fine suspendsuspend Injects fine after Clogs/pancakes Withdraws settling nicelyPancakes on injection PBS + 1% Sticks to tube Mannitol Injects fineinitially, then visible clumping PBS + 0.5% Difficult to suspend CMCSticks to tube Injects fine Injects fine after settling PBS + 0.5%Difficult to suspend CMC + 0.5% Injects fine SDS Injects fine aftersettling PBS + 0.5% Difficult to suspend Difficult to CMC + 0.02%Clogged on suspend Tween₂₀ withdrawal Pancake Pancakes on formed oninjection injection

TABLE 3 >45 μm 45-38 μm 38-25 μm <25 μm (Lot 077-132) (Lot 077-132) (Lot077-132) (Lot 077-110) 100 100 200 100 200 100 200 mg/ml mg/ml mg/mlmg/ml mg/ml mg/ml mg/ml PBS clumps clumps PBS clumps clumps 0.05% SDSPBS Can not Injects Injects Injects Injects Injects  0.5% draw into wellwell well well well SDS syringe Even w/ Even w/ Even w/ Even w/ settlingsettling settling settling PBS, Injects Injects 0.02% well well Tween20PBS clumps   1% Mannitol

Example 7 In Vitro Release of Pegaptanib Microspheres

The microspheres were analyzed to determine the in vitro release profileof pegaptanib. In vitro release of pegaptanib from microsphereformulations was determined in PBS (pH 7.4) containing 0.02% Tween-20and 0.05% sodium azide. Typically 10 mg of microspheres were added to 1mL buffer in a capped tube and placed in a shaking (150 cycles perminute) water bath incubator at 37° C. The release medium supernatantwas sampled periodically and assayed for conjugate quantity andapproximate purity by the reverse phase HPLC. The “burst release” wasdetermined by the percentage of drug that was released in the firstthree hours of incubation.

The release profiles were characterized by a low initial burst releaseof pegaptanib in the first 24 hours of release followed by a period ofsustained release ranging from 40 days to greater than 200 daysdependent on the composition and inherent viscosity of the PLGA polymerused to prepare the formulation.

Example 8 In Vivo Release of Pegaptanib Microspheres

The in vivo duration of release for microsphere formulations producedaccording to Example 1 were assessed in New Zealand rabbits bymonitoring blood plasma concentration after bilateral intravitreousdosing of the test formulations.

Microspheres containing pegaptanib were suspended in PBS injectionvehicle containing 0.02% surfactant at a concentration of 100 mgmicrospheres per milliliter. A volume of 50 μL of the test formulationsuspension was injected intravitreously into the eye using a 300 μLinsulin syringe fitted with a 29G, half-inch needle to provide a 5 mgdose of test formulation

Blood plasma samples were harvested at specified intervals and stored at−20° C. until analysis. Samples were analyzed for pegaptanibconcentration via Dual Hybridization-PCR Assay.

Animals

Adult male New Zealand White rabbits, weighing approximately 2.5-4.0 kgwere used.

Experimental Methodology

Animals were weighed and anesthetized with xylazine (5 to 10 mg/kgadministered subcutaneously) followed by ketamine (35 to 50 mg/kgadministered intramuscularly). One to two drops of Tropicamide® wereadministered to each eye prior to ophthalmic examination and dosing.

The intravitreal injections were administered on Study Day 1 by a boardcertified veterinary ophthalmologist (DACVO). For each of theintravitreal injections, the rabbit was placed in a lateral recumbentposition and the eye was topically anesthetized with 1 to 2 drops of0.1% proparacaine solution. The dose volume (for each eye) was 50 μLcontaining 5 mg of PLGA formulation. The test article was injectedintravitreously using a 300 μL insulin syringe fitted with a 29G,half-inch needle, or other appropriately sized needle. The needle wasinserted 1 to 2 mm posterior to the limbus in the superotemporalquadrant. The bevel was kept in an anterior position and the needle wasadvanced into the mid-vitreous. Antibiotic ointment (triple antibioticor equivalent) was administered to the eye, following injection. Theprocedure was repeated on the opposite eye. The first day of dosing wasdesignated as Study Day 1. Following dose administration on Study Day 1,the animals were observed for the duration of the study.

At the designated time point 2 mL of whole blood was collected from thelateral ear vein using potassium EDTA as the anti-coagulant. All sampleswere analyzed for pegaptanib concentrations via a dual hybridization-RTPCR Assay. The trapezoid rule method was used to calculate AUC andassess the relative bioavailability of pegaptanib release from PLGAmicrospheres. After the final blood collection, all animals wereeuthanized via anesthesia by ketamine:xylazine mixture followed by anoverdose of sodium pentobarbital.

Statistical Analysis

Statistical analysis of dual hybridization-RT PCR data was performedusing Graph Pad Prism® (GraphPad Software, Inc., San Diego, Calif.).Standard curve sample concentrations was calculated by a 4 parametercurve fit meeting appropriate statistical parameters.

Previous studies have demonstrated that after bilateral IVTadministration of pegaptanib sodium in a phosphate buffered salinesolution, pegaptanib vitreous and plasma concentrations decline in awell defined and predictable manner. The terminal phase rate constantwas equivalent in both vitreous humor and plasma and reflects the slowabsorption of pegaptanib from the eye into the systemic circulationafter an IVT injection. Thus blood plasma levels were used as a markerfor vitreous levels after intravitreous dosing of pegaptanib sodium inphosphate buffered saline.

It was expected that pegaptanib would be released in a sustained fashionfrom the test formulations into the vitreous and that the pegaptanibreleased into the vitreous would have distribution and clearanceproperties similar to the properties of pegaptanib dosed in a phosphatebuffered saline solution. Thus, the plasma pharmacokinetics ofpegaptanib reflected the in vivo release of pegaptanib from thesustained release formulation as elimination from the eye into systemiccirculation. This was considered to be the rate-limiting stepdetermining pegaptanib's plasma pharmacokinetics.

Example 9 Release Profiles of Pegaptanib Microspheres

(Formulations 093-063, 093-003-1, and 093-059)

Pegaptanib microspheres having a core load between about 12 and 15 w/w %were prepared as set forth in Example 1. Formulations 093-063,093-003-1, and 093-059 are summarized in Table 4. The formulations weresieved to provide microspheres having a particle size of less than 45μm.

TABLE 4 Drug Coreload 1-Day Burst Lot No. (wt. %) PLGA Polymer (%)093-033-1 14.3 50:50 4A 8.1 093-059 12.9 75:25 2A 13.5 093-063 14.450:50 3A 6.1

The in vitro release profile of the microspheres were analyzed as setforth in Example 7. The results of the in vitro release analysis aredepicted in FIG. 13. The results shown in FIG. 13 demonstrate thesustained release properties of the microparticles of the presentinvention. The results also demonstrate that the microparticles of thepresent invention can be selectively designed to control the release ofa biologically active agent over a desired time period. Preparingmicrospheres having various polymers demonstrated that the in vitrorelease of pegaptanib can be extended.

The in vivo release profile of the microspheres were analyzed as setforth in Example 8. The animal groups and blood collection schedule aresummarized in Table 5.

Group Identification and Sampling Schedule

TABLE 5 Pegaptanib Number Number of Total Content of Eyes/ Animals/Number of Group Test Article (w/w %) Route Timepoints TimepointTimepoint Animals Group Pegaptanib 14.4% Intravitreous 2 and 6 12 6 6 1MMicrospheres hours post 093-063 dose; and Study Days 2, 4, 9, 16, 23,30, 37, 44, 51, 58, 65, 72, 79, and 86 Group Pegaptanib 14.3%Intravitreous 2 and 6 12 6 6 2M Microspheres hours post 093-003-1 dose;and Study Days 2, 4, 9, 16, 23, 30, 37, 44, 51, 58, 65, 72, 79, and 86Group Pegaptanib 13.9% Intravitreous 2 and 6 12 6 6 3M Microsphereshours post 093-059 dose; and Study Days 2, 4, 9, 16, 23, 30, 37, 44, 51,58, 65, 72, 79, and 86

The in vivo release profile of the microspheres are illustrated in Table6 and FIGS. 14 and 15. Pharmacokinetic data is presented in Table 7.

TABLE 6 Formulation 093-003-1 093-059 093-063 Polymer 50:50-4A 75:25-2A50:50-3A Drug Content 14.2% 12.9% 14.4% API Dose (ug) 134 122 136 DayAve (ng/mL) Std Dev Ave (ng/mL) Std Dev Ave (ng/mL) Std Dev 0.00 0.000.00 0.00 0.00 0.00 0.00 0.08 0.67 0.17 0.85 1.06 0.72 0.66 0.25 1.990.82 3.57 2.67 1.61 1.04 2 3.38 1.26 5.00 1.42 4.57 1.91 4 4.72 1.443.41 1.65 5.41 2.65 9 2.24 0.56 2.14 1.30 2.67 0.98 16 1.08 0.32 1.390.69 0.94 0.32 23 0.50 0.30 0.60 0.47 0.55 0.13 30 4.81 1.71 4.60 1.731.90 0.38 37 4.15 2.09 5.61 1.69 5.94 1.22 44 1.93 0.85 2.62 0.90 5.522.16 51 0.74 0.50 0.84 0.53 3.29 1.80 58 0.20 0.21 0.28 0.19 1.14 0.9565 0.00 0.00 0.03 0.08 0.42 0.31 72 0.00 0.00 0.00 0.00 0.17 0.24 790.00 0.00 0.00 0.00 0.04 0.09 86 0.00 0.00 0.00 0.00 0.00 0.00

FIGS. 14 and 15 show plasma concentrations resulting from an 86 day invivo ocular study in rabbits dosed intravitreously with 5 mg of PLGAmicroparticles containing 15% weight percent pegaptanib are shown inFIGS. 14 and 15. FIG. 14 illustrates that three tested formulations allhad low in vivo burst release followed by a period maintenance of bloodplasma levels for 60-70 days. The in vitro release analysis of theseformulations indicated similar release profiles for the three testformulations which was predictive of the in vivo performance.

FIG. 14 illustrates that the three tested formulations had in vivorelease profiles that are well correlated with the in vitro releaseprofiles for ranking burst and duration of release.

The plasma concentration curves demonstrate a controlled burst releasethat was well predicted by in vitro release analysis. In addition, atypical PLGA microsphere release profile is observed with a lag phasefollowed by polymer controlled secondary release phase resulting in asustained plasma concentration above what is achieved by intravitreousdosing of the same dose of liquid pegaptanib sodium in rabbits. Thebioanalytical analysis revealed that the blood plasma concentration ofpegaptanib was sustained over a period of several weeks.

TABLE 7 Dose AUC_(tot) In vitro In vivo Formulation Description (ug)ng*hr/mL F_(rel) Burst Burst Liquid 141 4427 100% 093-003-1 50:50-4A 1343173  75%  8.08%  7.20% 093-059 75:25-2A 122 3589  94% 13.54% 12.93%093-063 50:50-3A 136 4429 104%  6.06%  5.83%

Example 10 Release Profiles of Pegaptanib Microspheres

(Formulations 093-023, 093-051, 077-189 and 093-041)

Pegaptanib microspheres having a core load between about 13 and 17 w/w %were prepared as set forth in Example 1. Formulations 093-023, 093-051,077-189 and 093-041 are set forth in Table 8. The formulations weresieved to provide microspheres having a particle size of less than 45μm.

TABLE 8 Drug Coreload 1-Day Burst Lot No. (wt. %) PLGA Polymer (%)093-023 13.6 50:50 5A 32.8 093-051 14.1 65:35 3A 3.4 077-189 16.1 75:254A 5.2 093-041 13.6 PLA 0.36 dL/g IV 1.6

The in vitro release profile of the microspheres were analyzed as setforth in Example 7. The results of the in vitro release analysis of aredepicted in FIG. 16. The results shown in FIG. 16 demonstrate thesustained release properties of the microparticles of the presentinvention. The results also demonstrate that the microparticles of thepresent invention can be selectively designed to control the release ofa biologically active agent over a desired time period. Preparingmicrospheres having various polymers demonstrated that the in vitrorelease of pegaptanib can be extended.

The in vivo release profile of the microspheres were analyzed as setforth in Example 8. The in vivo release profile of the microspheres areillustrated in Table 9 and FIG. 17. Pharmacokinetic data is presented inTable 10.

TABLE 9 Formulation 093-023 093-051 077-189 093.041 Polymer 50:50-5A65:35-3A 75:25-4A PLA (0.38) Drug Content 13.6% 14.1% 16.1% 13.6% APIDose (ug) 128 133 152 128 Day Ave (ng/mL) Std Dev Ave (ng/mL) Std DevAve (ng/mL) Std Dev Ave (ng/mL) Std Dev 0 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.08 0.79 0.40 0.15 0.30 0.69 0.97 0.25 0.33 0.25 2.59 1.130.60 0.26 1.78 1.12 1.45 0.96 1 5.83 3.67 1.91 0.25 2.81 0.96 3.78 2.413 3.23 1.84 1.20 0.32 2.38 1.09 2.73 1.38 7 2.55 0.61 1.02 0.28 2.471.36 1.86 0.73 14 1.64 0.65 1.45 0.39 1.23 0.31 1.21 0.10 21 0.99 0.350.73 0.22 0.72 0.31 0.60 0.17 28 3.43 0.92 0.46 0.11 0.46 0.04 0.22 0.1835 7.28 2.10 3.24 2.22 0.25 0.05 0.00 0.00 42 3.06 0.94 6.96 2.45 0.250.07 0.00 0.00 49 1.50 0.52 5.63 1.38 0.38 0.09 0.00 0.00 56 0.60 0.172.63 1.03 1.25 0.50 0.00 0.00 63 0.11 0.12 0.56 0.18 2.75 0.82 0.00 0.0070 0.03 0.06 0.26 0.14 3.64 1.33 0.00 0.00 77 0.00 0.00 0.00 0.00 3.161.54 0.00 0.00 84 0.00 0.00 0.00 0.00 1.53 0.83 0.00 0.00 91 0.00 0.000.00 0.00 0.78 0.35 0.00 0.00 98 0.00 0.00 0.00 0.00 0.35 0.16 0.00 0.00105 0.00 0.00 0.00 0.00 0.14 0.22 0.00 0.00 112 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 119 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 126 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 133 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 140 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 147 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 154 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 161 0.000.00 0.00 0.00 0.00 0.00 0.03 0.08 168 0.00 0.00 0.00 0.00 0.00 0.000.05 0.13 175 0.00 0.00 0.00 0.00 0.00 0.00 0.18 0.28 182 0.00 0.00 0.000.00 0.00 0.00 0.47 0.53 196 1.44 0.34 210 1.71 0.72 217 1.25 0.48 2240.93 0.47 231 0.62 0.24 238 0.31 0.15 245 0.18 0.16 252 0.12 0.15 2590.00 0.00

FIG. 17 depicts plasma concentrations resulting from an 8-month in vivoocular study in rabbits dosed intravitreously with 5 mg of PLGAmicroparticles containing 15% weight percent pegaptanib. Of note is theabsence of a large burst release common to PLGA formulations containingwater soluble or hydrophilic compounds. Blood plasma levels weremonitored as a surrogate marker for vitreous concentrations based on theestablished pharmacokinetics of Pegaptanib Sodium.

FIG. 17 illustrates that the four tested formulations all had low invivo burst release followed by a period maintenance of blood plasmalevels. The in vitro release analysis of these formulations indicatedsimilar release profiles for the three test formulations which waspredictive of the in vivo performance. FIG. 17 illustrates that the fourtested formulations had in vivo release profiles that are wellcorrelated with the in vitro release profiles for ranking burst andduration of release.

The plasma concentration curves demonstrate a controlled burst releasethat was well predicted by in vitro release analysis. The bioanalyticalanalysis revealed that the blood plasma concentration of pegaptanib wassustained over a period of several months.

TABLE 10 Dose AUC_(tot) In vitro In vivo Formulation Description (ug)ng*hr/mL F_(rel) Burst Burst Liquid 141 4427 100% 093-023 5050-5A 1283924  98% 11.9% 21.1% 093-051 6535-3A 133 3974  96%  3.4%  6.9% 077-1897525-4A 151 3449  73%  5.2% 10.2% 093-041 PLA 0.36 128 2573  64% 16.2%13.7%

Example 11 In Vivo Release of Pegaptanib-Loaded Microparticles

(Formulations 079-089 and 079-102)

Applicants evaluated the in vivo release properties of pegaptanib-loadedmicroparticles. Applicants administered pegaptanib-loadedpoly(lactide-co-glycolide) (PLGA) microspheres, formed by the process asset forth in Example 1, intravitreously in New Zealand White rabbits andplasma samples were collected at various time points from 2 hours to 28days as set forth in Table 11. This evaluation demonstrated thefeasibility of delivering pegaptanib over a period of approximately onemonth or more from a (PLGA) polymer based sustained release formulation.

TABLE 11 Number of Number of animals/time Total Number of Group Timepoints point animals Intravitreous Injection 6 3 18 of MicrospheresControl Intravitreous 2 3 6 Injection of Blank Microspheres ControlIntravitreous 2 3 6 Injection of 50 μL 0.9% Sodium Chloride Total Numberof 30 AnimalsMaterials and Methods

PLGA microsphere formulations 079-089 and 079-102 were prepared as setforth in Example 1 above and were blended to provide sufficient materialfor animal dosing. Microspheres containing pegaptanib 15% on a weightpercent basis were suspended in PBS injection vehicle containing 0.02%surfactant at a concentration of 100 mg microspheres per milliliter. ThePegaptanib-loaded PLGA microsphere formulations were administered byintravitreous injection (IVT) in New Zealand White rabbits. Placebomicrospheres were dosed in an identical manner in control. Blood Plasmasamples were collected at various time points, from 2 hours to 28 daysas set forth in Table 10. Samples were stored at −20° C. until analysis.Samples were analyzed for pegaptanib concentration via a DualHybridization-PCR assay as set forth in Example 14. (see PatentApplication Publication No. WO 2006/012468, which is incorporated hereinby reference in its entirety).

Animals

Adult female New Zealand White rabbits, weighing approximately 2.5-4.0kg were used. The groups are summarized in Table 11. The treatmentschedule is summarized in Table 12.

Treatment Schedule

TABLE 12 Number Number of of Eyes/ Animals/ Total Number Group TestArticle Route Timepoints Timepoint Timepoint of Animals Group 1Pegaptanib Intravitreous 2 hour, 1, 5, 6 3 18 Microspheres 9, 14, 28 dayGroup 2 Blank Intravitreous 1, 9 day 6 3  6 MicrospheresExperimental Methodology

Intraocular pressure was measured in each animal prior to anesthesiausing a hand-held applanation tonometer (Tonopen™). Animals were weighedand anesthetized with ketamine/xylazine administered intramuscularly.Tropicamide® were administered to each eye prior to ophthalmicexamination. With the rabbit in right lateral recumbency, the left eyewas topically anesthetized with 0.1% proparacaine solution.

A volume of 50 μL of the test article was injected intravitreously,using a 500 μL insulin syringe fitted with a 29G, half-inch needle, orother appropriately sized device. For Intravitreous injections, theneedle was inserted 1-2 mm posterior to the limbus in the superotemporalquadrant. The bevel was kept in an anterior position and the needle wasadvanced into the mid-vitreous. Antibiotic ointment (triple antibioticor equivalent) was administered to the eye following injection. Theprocedure was repeated on the right eye. Observations were recorded,including, but not limited to, leakage of test material from theinjection site.

Whole blood (500 μL) was collected from the lateral ear vein. Foranimals in Group 1 (Day 28 time point), blood samples were collected atthe following time points: 2 and 6 hours, 1, 3, 5, 9, 14, and 21 days,in addition to the terminal time point.

Statistical Analysis

Statistical analysis was performed using Graph Pad Prism. Samples areanalyzed by 4 parameter curve fit meeting appropriate statisticalparameters.

Results

Plasma concentrations resulting from a 28 day in vivo oculardistribution study in rabbits dosed intravitreously with 5 mg of PLGAmicroparticles containing 15% weight percent pegaptanib are shown inTable 13 and FIG. 12. The results show that the microparticles have alow burst release. A large burst release, common to PLGA formulationscontaining water soluble or hydrophilic compounds, is absent. Inaddition plasma pegaptanib concentration levels are measured at arelatively constant level between 0.05-0.4 nM over the 28 day studyperiod relative to an equivalent IVT liquid pegaptanib dose, indicatinga sustained release of pegaptanib in the vitreous was achieved.

It is known in the art that modifying the polymer composition of asustained release microparticle formulation affects the rate of polymerdecomposition in vivo and therefore effects the release characteristicsof the microparticle formulation. Therefore demonstrating a releaseduration of one month from a microsphere formulation in vivo indicatesthat a release duration of greater than one month from a microsphereformulation in vivo is feasible.

TABLE 13 Pegaptanib Plasma Concentration Following IntravitreousInjection Pegaptanib Microsphere Pegaptanib Liquid Days Conc (nM) SDDays Conc (nM) StDev 0.08 0.055 0.078 0.08 0.051 0.05 0.25 0.142 0.2000.25 0.436 0.12 1 0.126 0.065 1 1.861 0.06 4 0.137 0.067 5 1.476 0.26 70.080 0.014 8 0.941 0.04 11 0.051 0.005 11 0.539 0.21 16 0.069 0.016 150.451 0.13 19 0.138 0.070 19 0.241 0.06 23 0.362 0.203 23 0.219 0.09 260.423 0.273 28 0.088 0.02 28 0.415 0.271

Example 12 Release Profile of Pegaptanib Microparticles

(Water-in-Oil-in-Water)

Table 14 shows the properties of pegaptanib microparticles that wereprepared as set forth in Example 2. The in vitro release profiles of thepegaptanib microparticles were determined by the process as set forth inExample 7

The release profiles of the microparticles from Table 14 are shown inFIG. 10. FIG. 10 is a graph depicting in vitro dissolution rate profilesdemonstrating control release kinetics from Pegaptanib-PLGAmicrospheres.

TABLE 14 EYE001 Core Load % Encapsulation Particle Surface DescriptionYield (% w/w) Efficiency Size Morphology  EYE001 50:50 PLGA 65.78% 7.09± 0.84 46.14 ± 5.5  <10 μm Smooth  EYE001 50:50 PLGA 66.02% 7.05 ± 0.0747.09 ± 0.45 <10 μm Smooth  EYE001 50:50 PLGA 66.28% 8.22 ± 0.09  52.8 ±0.54 NT NT Placebo 50:50 PLGA 64.05% n/a n/a <10 μm Smooth  EYE001 50:50PLGA 56.49% 9.02 ± 0.11 59.08 ± 0.69 <10 μm Smooth Placebo 50:50 PLGA64.10% n/a n/a <10 μm Smooth  EYE001 50:50 PLGA 87.01% 7.70 ± 0.46 51.06± 3.04 <10 μm Smooth

The results of the in vitro release analysis of are depicted in FIG. 10.FIG. 10 shows the release properties of microparticles formed by thew/o/w process as set forth in Example 2. The results shown in FIG. 10demonstrate the sustained release properties of the microparticles ofthe present invention. The results also demonstrate that themicroparticles of the present invention can be selectively designed tocontrol the release of a biologically active agent over a desired timeperiod. Preparing microspheres having various polymers demonstrated thatthe in vitro release of pegaptanib can be extended.

Example 13 Pegaptanib Inhibition of VEGF Induced Tissue FactorExpression in HUVEC Cells

Pegaptanib microspheres were prepared as set forth in Example 1 above.The microspheres were analyzed to determine if pegaptanib maintained itsefficacy following release from the microspheres.

HUVEC cells were plated at 1.5×10⁵ cells/well in complete medium(Cascade Biologics Medium 200, supplemented with Low Serum GrowthSupplement and Penicillin, Streptomycin, and Amphotericin B (PSA)) in 24well plates and allowed to attach overnight in a 37° C./5% CO₂incubator. Sixteen hours later, complete media was removed and cells arewashed once with 1% medium (Cascade Biologics Medium 200, supplementedwith 1% Fetal Bovine Serum and PSA). Cells were then starved in the 1%medium for 4 hours in a 37° C./5% CO₂ incubator. During starvation,assay controls and samples were prepared. Assay controls included 1%medium alone (zero control), 1% medium with 12.5 ng/mL VEGF (VEGFinduction control), and 1% medium with Pegaptanib at 10 nM (Pegaptanibcontrol). The test microsphere samples were prepared at a concentrationof 10 nM in 1% medium with VEGF (12.5 ng/mL). After 4 hours ofstarvation, media was removed and the prepared assay samples were addedto respective wells. All controls and test samples were done induplicate (2 wells each). Cells were treated for 1 hour in a 37° C./5%CO₂ incubator. After the 1 hour incubation, media was removed and cellswere washed with sterile 1×PBS. Cells were then lysed with RLT/βME lysisbuffer (Qiagen). Lysed cells were collected in sterile tubes and storedat −80° C. or used immediately for total RNA isolation. RNA isolationwas performed using the Qiagen RNeasy® Protocol. cDNA was made and theTissue Factor gene was quantitated using a typical Taqman® Real Time PCRprotocol.

The results of cell proliferation assays of human umbilical veinendothelial cells (HUVEC) incubated with EYE001 formulations afterrelease from PLGA microparticles are shown in FIG. 11. The figureillustrates that addition of VEGF to the media increased expression ofTissue Factor by 9-fold. In the presence of 10 nM pegaptanib, TissueFactor expression was reduced to basal levels. Pegaptanib released fromPLGA microspheres had a similar effect on Tissue Factor expression asnative pegaptanib. The results thereby demonstrated that the in vitrobioactivity of pegaptanib was unchanged by the microsphere fabricationprocess and subsequent in vitro release.

Example 14 Dual Hybridization-PCR Analysis of in Vivo Pegaptanib Release

Vitreous Digestion

Rabbit microsphere Vitreous samples are placed in 15 mL and 50 mL tubesand incubated overnight at 37° C. Following the incubation, Proteinase K(20 μL/sample) is added and the samples are incubated for 2 hours at 65°C. After 2 hours the samples are cooled to room temperature and then putin ice. The cooled samples are ready for dual hybridization assay.Plasma does not require digestion. 1×PBS and SDS is not added to theVitreous digestion

The digestion cocktail is prepared (per sample) as follows:

1xPBS 500 μL Hyaluronidase cocktail  20 μL Blendzyme III  20 μL SDS  5μL DNAse  10 μL

Hyaluronidase cocktail is a mixture of 4 hyaluronidases in equal parts 5μL. Each Hyaluronidase cocktail is a 1 mg/mL solution in 1×PBS and1×BSA. Blendzyme (Roche Diagnostics Corporation Indianapolis, Ind.);Liberase Blendzymes are mixtures of highly purified collagenase andneutral protease enzymes, formulated for efficient, gentle, andreproducible dissociation of tissue from a wide variety of sources) ismade as 7 mg/ml solution in 1×PBS. Proteinase K is made as 20 mg/mlsolution in 1×PBS.

Dual Hybridization Assay

(Plasma)

Standards were prepared in 1× tissue (control tissue diluted 1:10). 165μL of primer mix was added per sample into 0.2 PCR tubes. Primer mix wasprepared by adding 1 μM detection primer to Hybridization buffer so thatthe final concentration of the detection primer in the required mixturewas 1 nM. Standards or sample (25 μL) were added. Capture beads (5 μL)were added. The PCR tubes were then placed in a Thermal Cycler andprogrammed to run 75° C. for 15 minutes, 37° C. for 1 hour and then downto 25° C. Then the samples and standards were transferred from the PCRtubes into 96 well plates and 180 μL were transferred from each.

The plate was then run in a Kingfisher® 96 magnetic particle processor.

The following 96-well plates were prepared:

-   -   1-96 well plate (100 μL of water in each well)    -   5-96 well plates (200 μL of 1× wash buffer in each well)

The magnetic comb picked up the beads and moved them from plate toplate. The beads were washed in each plate leaving behind anything thatwas not specifically bound to the beads. The beads were deposited in thewater plate at the end of the run. At this point the plate was ready tobe run in Taqman® RT PCR.

Taqman® RT PCR

A 384-well plate was used. Total volume in a well was 10 μL. (Sample=4μL; Cocktail=6 μL). A duplicate was prepared for each sample in the 96well plate.

The following cocktail was prepared per 384 well:

H20  0.4 μL 2xPCR mix   5 μL Forward primer 0.05 μL Reverse primer 0.05μL Probe  0.5 μL

First the cocktail was added and then the samples from the 96 well platewere transferred. The 384 well plate was spun in a centrifuge (1000 rpm)for less than a minute. The plate was then run in Taqman: 50° C. for 2min, 95° C. for 10 min, and then 40 cycles of 95° C. 15 seconds to 60°C. for min.

Data Analysis

At the end of the run, data was available for the run from the SDSsoftware. These data were then plugged in Microsoft Excel and Prism andthe concentrations of the samples were then extrapolated from a standardcurve.

The results of a 28 day in vivo release study of pegaptanib from PLGAmicroparticles are discussed in Example 11. FIG. 12 is a graph showingpegaptanib concentration in rabbits plasma samples dosed intravitreousor subconjunctival with 5 mg of PLGA microparticles containing 15%weight percent pegaptanib.

Example 15 Delayed Release Microparticle Pegaptanib Dosing Regimen

Step 1.

Administer a 100 μL pharmaceutical formulation comprising a bolus ofabout 0.3 mg free pegaptanib in solution and delayed release PLGAmicroparticles encapsulating about 35 mg pegaptanib.

Step 2.

The microspheres will have an initial burst of about 5-30% of pegaptaniband then will release at some rate constant over a predefined period.

Step 3.

At the end of the microsphere release profile, a second burst will occurreleasing a second bolus of pegaptanib bringing the vitrealconcentration to about 0.3 mg.

Step 4.

Four weeks post burst, during which time the polymeric metabolites arecleared, a new pegaptanib/microparticle injection would be administeredas described in Step 1.

INCORPORATION BY REFERENCE

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. All patents,patent applications, and published references cited herein are herebyincorporated by reference in their entirety.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

We claim:
 1. A sustained release suspension composition for intravitrealadministration consisting essentially of a plurality of microparticlessuspended in a pharmaceutically acceptable liquid carrier comprisingphosphate buffer saline (PBS) and a surfactant comprising sodium dodecylsulfate (SDS), wherein the liquid carrier is suitable for intravitrealadministration, wherein said microparticles comprise: (a) a biologicallyactive anti-VEGF aptamer; and (b) poly(lactic acid) (PLA) or poly(lacticacid-co-glycolic acid) (PLGA) polymer and have a smooth, non-pittedexternal morphology, wherein said polymer has a monomer ratio oflactide:glycolide in the range of about 40:60 to 100:0; said suspensioncomprises 100-300 mg microparticles per mL of said liquid carrier; saidmicroparticles release said biologically active anti-VEGF aptamer over aperiod of at least 40 days; said microparticles have a particle sizedistribution in the range of 10 μm to 45 μm in diameter; saidmicroparticles comprise a PLA or PLGA polymeric material incorporating acore load of at least 7 wt % of said anti-VEGF aptamer and; saidmicroparticles release said anti-VEGF aptamer at an initial burst ofless than 15 wt % of said core load within 24 hours of administration.2. The composition of claim 1, wherein the microparticles release theanti-VEGF aptamer over a period of at least 3 months.
 3. The compositionof claim 1, wherein the microparticles release the anti-VEGF aptamerover a period of about 3-6 months.
 4. The composition of claim 1,wherein the microparticles are syringable through a 29-gauge needle ornarrower.
 5. The composition of claim 1, wherein the microparticles havea mean diameter of about 30 μm.
 6. The composition of claim 1, whereinthe microparticles have a mean diameter of about 15 μm.
 7. Thecomposition of claim 1, wherein the microparticles are syringablethrough a 27-gauge needle or narrower and have a diameter of less thanor equal to 75% of the inner diameter of the needle.
 8. The compositionof claim 1, wherein the microparticles are syringable through a 27-gaugeneedle or narrower and have a diameter of less than or equal to 50% ofthe inner diameter of the needle.
 9. The composition of claim 1, whereinthe microparticles are syringable through a 27-gauge needle or narrowerand have a diameter of less than or equal to 25% of the inner diameterof the needle.
 10. The composition of claim 1, wherein themicroparticles have a core load of at least 10% by weight.
 11. Thecomposition of claim 1, wherein the microparticles have a core load ofat least 15% by weight.
 12. The composition of claim 1, wherein themicroparticles have a core load of at least 20% by weight.
 13. Thecomposition of claim 1, wherein the microparticles exhibit a 24 hourburst of less than 10 wt % of said core load.
 14. The composition ofclaim 1, wherein the microparticles exhibit a 24 hour burst of less than5 wt % of said core load.
 15. The composition of claim 1, wherein themicroparticles form a homogeneous or un-agglomerated suspension with thepharmaceutically acceptable carrier.
 16. The composition of claim 1,wherein the microparticles are microspheres.
 17. The composition ofclaim 1, wherein the internal morphology of the microparticles ishomogeneous or monolithic.
 18. The composition of claim 1, wherein theanti-VEGF aptamer is a therapeutic agent suitable for the treatment ofan ophthalmic disease or disorder.
 19. The composition of claim 18,wherein the anti-VEGF agent is conjugated to a non-toxic, long-chain,hydrophilic, hydrophobic or amphiphilic polymer.
 20. The composition ofclaim 19, wherein the anti-VEGF aptamer is conjugated to polyethyleneglycol.
 21. The composition of claim 20, wherein the anti-VEGF aptameris pegaptanib.
 22. The composition of claim 21 wherein pegaptanib isreleased from the microparticles at a rate ranging from about 0.01 toabout 10 micrograms (μg) per day.
 23. The composition of claim 22wherein pegaptanib is present in an amount sufficient to providepegaptanib plasma concentrations of about 0.05-0.40 nM throughout anadministration period of at least 3 weeks.
 24. The composition of claim21 wherein the microparticles are suspended in a pegaptanib solution.25. The composition of claim 1 wherein the liquid carrier furthercomprises a surfactant selected from the group consisting of poly(vinylalcohol), carboxymethyl cellulose, lecithin, gelatin, poly(vinylpyrrolidone), polyoxyethylenesorbitan fatty acid esters and mannitol.26. The composition of claim 25 wherein the surfactant is selected frompolyoxyethylenesorbitan fatty acid esters and mannitol.
 27. A sustainedrelease suspension composition consisting essentially of a plurality ofmicroparticles having a smooth, non-pitted external morphology suspendedin a pharmaceutically acceptable liquid carrier comprising phosphatebuffer saline (PBS) and a surfactant comprising sodium dodecyl sulfate(SDS), wherein the liquid carrier is suitable for intravitrealadministration, wherein said microparticles form a homogeneous orun-agglomerated suspension with the pharmaceutically acceptable carrier,said suspension comprises 100-300 mg microparticles per mL of saidliquid carrier; wherein said microparticles comprise a poly(lacticacid-co-glycolic acid) (PLGA) polymeric material having a monomer ratioof lactide:glycolide of 75:25 and incorporating a core load of at least7 wt % of pegaptanib, wherein said microparticles have a particle sizedistribution in the range of 10 μm to 45 μm in diameter, wherein saidmicroparticles release pegaptanib over a period of at least 40 days atan initial burst of less than 15 wt % of said core load within 24 hoursof administration, wherein pegaptanib is released from themicroparticles at a rate ranging from about 0.01 to about 10 micrograms(μg) per day, and wherein said microparticles are syringable through a27-gauge needle or narrower.
 28. The composition of claim 27 whereinpegaptanib is released from the microparticles at a rate ranging fromabout 0.1 to about 6 μg per day.
 29. The composition of claim 27 whereinpegaptanib is released at a rate sufficient to achieve pegaptanib plasmaconcentrations of about 0.05-0.40 nM throughout an administration periodof at least 3 weeks.
 30. The composition of claim 27 wherein pegaptanibis released at a rate sufficient to achieve pegaptanib plasmaconcentrations of about 0.05-0.40 nM throughout an administration periodof at least 6 weeks.
 31. The composition of claim 27, wherein themicroparticles release pegaptanib over a period of at least 3 months.32. The composition of claim 27, wherein the microparticles have a coreload of at least 10% by weight.
 33. The composition of claim 27, whereinthe microparticles exhibit a 24 hour burst of less than 10 wt % of saidcore load.
 34. The composition of claim 27, wherein the microparticlesexhibit a 24 hour in vitro burst of less than 5 wt % of said core load.35. The composition of claim 27, wherein the carrier further comprises apharmaceutically acceptable surfactant or excipient.
 36. The compositionof claim 1, wherein the microparticles release the anti-VEGF aptamerover a period of at least 180 days.
 37. The composition of claim 1,wherein the microparticles release the anti-VEGF aptamer over a periodof at least 365 days.
 38. The composition of claim 27, wherein themicroparticles release the anti-VEGF aptamer over a period of at least180 days.
 39. The composition of claim 27, wherein the microparticlesrelease the anti-VEGF aptamer over a period of at least 365 days.